The complexity and unique characteristics of the Trypanosoma cruzi genome and the relative paucity of tools to manipulate that genome are some of the challenges in the study of parasite persistence and pathogenicity that results in Chagas disease. As the highest impact infectious disease of the Americas with up to 20 million humans and innumerable animals affected, T. cruzi is plagued by poor diagnostic tools, inadequate treatment options and no effective vaccines. New discoveries in all these areas would be greatly facilitated by improved genome editing tools for T. cruzi. We have recently applied CRISPR technology to T. cruzi and demonstrate dramatically increased gene knockout and gene insertion efficiency, providing an unparalleled opportunity for genetic analysis in this important human pathogen. In this project we will further enhance the CRISPR-Cas system in T. cruzi by improving the ability to regulate Cas9 expression, better defining the mechanisms of DNA recombination used to repair Cas9-mediated double-stranded breaks, and using this information to optimize homologous recombination in T. cruzi. Using the demonstrated multiplexing capabilities of CRISPR-Cas9, we will then assess the ability to mutate of large numbers of genes encoding trans-sialidase (ts) molecules in order to evaluate the hypothesis that the ts family of molecules serves an immune evasion function for T. cruzi. This proposal combines the novel use and further development of a powerful genetic system that we demonstrate to be highly efficient in T. cruzi, with an important biological question that up to no was not experimentally tractable. Understanding the role of large gene families in the persistence and pathology of T. cruzi is only possible if we can reduce the number of family members and observe the consequences. Completion of this work is also expected to firmly establish the CRISPR-Cas system as an indispensable tool for the study of T. cruzi.